The broad long-term objective of this proposal is to develop better therapeutics for acute myeloid leukemia (AML). There is an urgent unmet need for development of new therapy for AML as only 25% of patients achieving complete remission remain disease free. There is increasing evidence that an imbalance in the sjDhingolipid biostat contributes to pathogenesis and drug resistance in AML. Preliminary data demonstrate altered sphingolipid metabolism in this disease, including increased levels of pro-survival sphingosine-1-phosphate and constitutive overexpression of key enzymes, acid ceramidase (AC) and sphingosine kinase 1 (Project 3). Our central hypothesis is that AC inhibition leads to therapeutic efficacy by both increasing production of pro-apoptotic ceramide species and by blocking conversion of ceramide to pro- survival glycosylated metabolites. We will test the hypothesis that AC mediates survival of AML blasts/leukemia stem cells (LSC) in Specific Aim 1. Preliminary data indicate that AC is a valid therapeutic target in AML. AC activity is overexpressed in patient AML cells and in LSC; AC inhibition with LCL-204 leads to apoptosis of AML cells. We show that AC inhibition causes accumulation of pro-apoptotic ceramide, predominantly Cis species. Compelling preliminary data support a model in which S1P/AC/Mcl-1 axis regulates leukemia cell survival. We will also test the hypothesis that AC confers a drug resistance phenotype in AML (Specific Aim 2). We have found that AC expression/NF-KB activation modulates expression of P-glycoprotein (P-gp) and that AC inhibition sensitizes drug resistant AML cells to standard AML therapeutics. We suggest that P-gp-mediated drug resistance does not involve conventional efflux of therapeutics. Rather, based on Project 4 findings, we hypothesize that downregulation of P-gp blocks ceramide flux leading to decrease in levels of pro-survival ceramide metabolites. Our overall strategy will utilize a genetic approach to inhibit AC in both specific aims. We will also test therapeutic AC inhibitors LCL-204 and its analogs in murine LSC models. We expect that the proposed research will have significant impact on the field of AML biology. We anticipate that our in vivo work with AC inhibitors will validate a novel sphingolipid therapeutic approach for treatment of AML.